The heat sensor TRPV1 channel is a polymodal receptor that plays a key role in mediating neuronal pain caused by various noxious stimuli. One such stimulus is extracellular acidification caused by inflammation, tissue damage and ischemia. Low pH is thought to activate TRPV1 both directly by serving as a channel activator and indirectly by potentiating the channel's response to other stimuli. How TRPV1 activation is controlled by pH as well as its relation to activation by heat, ligands, and endogenous channel modulators remains largely unknown. Importantly, the highly unique susceptibility of TRPV1 activity to a variety of physical and chemical factors makes the channel an attractive target for clinical intervention of pain. The overarching goal of our research is to understand the cellular sensing function of TRPV1 by elucidating molecular mechanisms underlying its polymodal activation by heat, capsaicin and other stimuli. In the proposed study we aim to reveal the structural and mechanistic nature of extracellular H+ regulation of TRPV1. As H+-induced TRPV1 activity has heat-dependent and agonist-dependent components, this investigation will also shed light on how heat and agonist control TRPV1 activity. We approach our goal through a combination of optical, electrophysiological, and molecular methods. In particular, we will apply a patch fluorometry approach to directly observe structural changes in the channel protein or the binding of regulatory molecules, using fluorophores as molecular sensors. Simultaneous fluorescent and electrical recordings permit direct correlation of structural changes to their effects on channel activation. Using these methods, we will address questions on how changes in pH, temperature, and the concentration of agonists are sensed by TRPV1, what channel structures convey these stimuli, and how these stimuli converge to control TRPV1 activation. Answers to these questions should directly benefit the development of new clinical tools for treating TRPV1- mediated neuronal pain.